PROJECT SUMMARY/ABSTRACT. Since neurons are post-mitotic, long-lived, and extraordinarily large, maintaining the proteome is of particular importance. Not surprisingly, disturbances in protein homeostasis (?proteostasis?) have been associated with numerous neurodegenerative disorders, as well as aging. How protein turnover, is regulated in time and space is thus an essential cell biological question with profound implications for neuronal function and disease. Endosome transport in dendrites is not well understood. Our specific aims are to: Aim 1) discover how dendritic degradative flux (i.e. transport, acidification, fusion with lysosomes) is coordinated along dendrites by two small GTPases localized to late endosomes and lysosomes, Rab7 and Arl8b, Aim 2) identify the roles of two Rab7 effectors in regulating proteostasis, and Aim 3) discover functional consequences of disrupted endosomal maturation for neuronal health, (including degradation of membrane receptors, of aggregated proteins, maintenance of dendrites, and cell survival). Endosome maturation involves recruitment of small GTPases (Rab7 and Arl8b) and their effectors to regulate acidification, motility, and fusion events. Our recent work has implicated Rab7 in spatial regulation of degradative flux. Rab7 binds to multiple effectors, but how they spatially regulate endosome behavior in dendrites is poorly understood. Notably, Rab7 itself is mutated in Charcot-Marie-Tooth disease 2B and many Rab7 effectors are linked to diseases, highlighting the centrality of endosomal maturation pathways for normal neuronal function. Current barriers to progress include missing ?molecular handles? for studying the heterogeneity of late endosomes and lysosomes, lack of endogenous trackable cargos that can be used to follow degradation, and ignorance of the many endosomal effectors which regulate endosomal flux. The premise for this application rests on our own recent data: 1) Contrary to common belief, several endogenous dendritic receptors are not degraded in dendrites, but instead are retrogradely transported to the soma/proximal dendrite in a Rab7-dependent manner. Degradation thus does not take place in dendrites, but in somatic lysosomes. 2) LAMP1, the most commonly used lysosome marker in fibroblasts, is present in many compartments which are not degradative lysosomes, especially more distally in dendrites, highlighting neural-specific mechanisms. 3) Most Rab7-containing late endosomes in distal dendrites contain no LAMP1, and are thus different from late endosomes in fibroblasts which overwhelmingly contain both Rab7 and LAMP1. We refer to them as ?early? late endosomes to distinguish them from the conventional Rab7+/LAMP1+ late endosomes. These new discoveries allow us to now address a significant overarching question: How does the expansive dendritic arbor coordinate endosome maturation and degradation, and how do failures of proteostasis affect cellular function of neurons? Our long-term goal is to unravel the mechanisms of proteostasis in dendrites.